Fig 1: Top 21 ligands from CBkinUT binding to the protein PTPB. Scores are negative. Least negative score is the best.
Surrogate cloning was also completed with Joey's protein in mind. Bacteria cells grown were also sonicated. Next week, the protein will be harvested and a protein gel check will be used to confirm the correct protein was found and that it had not dimerized.
Week 12:
Good - Dr. B 11/19/12
Fig.1: Homology alignment of Mycobacterium Tuberculosis Protein Tyrosine Phosphatase PtpB with lmo1800 PTP in Listeria Monocytogenes
Fig. 2: Clashscores Multicriterion chart of Homology model template
Fig. 3: Multi-criterion kinemage of homology template with pink indicating bad overlap
Week 11:
After a few attempts to get the PNIC-Bsa4 at a high concentration, cloning was attempted. Only one colony of the bacteria grew after one day, but I will try cloning again to grow more colonies.
Fig. 1: DH5alpha bacteria with gene of interest plated with Kanamycin and 5% sucrose.
Fig. 2: GEL check of pNIC-Bsa4 cut with T4 polymerase
Week 10:
Fig. 1: PniC-Bsa4 nanodrop
Fig. 2: Pnic-Bsa4 concentration
This week I performed several MIDI preps for the pNIC-Bsa4 to yield a higher concentration in order to continue the cloning process. However, my first MIDI prep resulted in a low concentration due to someone switching the caps on the elution and wash buffer solution. My next MIDI prep resulted in a decent yield, so next week I will try to finish the cloning process using the 69.4 ng/uL concentration of pNIC-Bsa4. I also completed a PCR cleanup on the pNIC-Bsa4, but that only boosted my concentration by 15 ng/uL.
Week 9:
Fig 1: Nanodrop of my PNIC-BSa4 cut DNA
This low concentration shows that the procedure needs to be redone.
Since there were no large concentrations of the pNIC-Bsa4, new preparation using MIDI prep has to be completed. This will hopefully increase the concentration of the cut pNIC-Bsa4. From there, I can continue the cloning process.
Week 8:
102112 - good -- Dr. B
Virtual Screening Refresher was completed:
Fitness
S(hb_ext)
S(vdw_ext)
S(hb_int)
S(int)
Ligand name
81.65
5.62
60.65
0
-7.36
'9039798|CB5k_10|sdf|947|dock8'
81.56
6
54.08
0
1.21
'9040519|CB5k_10|sdf|1056|dock6'
81.26
13.11
53.2
0
-5.01
'9036564|CB5k_10|sdf|426|dock10'
81.26
4.54
59.33
0
-4.85
'9057940|CB5k_10|sdf|3998|dock2'
80.84
18.09
51.04
0
-7.43
'9038483|CB5k_10|sdf|723|dock6'
80.3
6.06
61.95
0
-10.94
'9037146|CB5k_10|sdf|529|dock1'
79.78
17.98
48.95
0
-5.50
'9035520|CB5k_10|sdf|251|dock7'
77.42
12
49.84
0
-3.12
'9059625|CB5k_10|sdf|4116|dock10'
77.1
1.74
55.37
0
-0.78
'9038770|CB5k_10|sdf|778|dock3'
76.89
12
54.62
0
-10.21
'9049544|CB5k_10|sdf|2822|dock4'
Table 1: Table of Top 10 Ligands through the use of GOLD.
Cloning was also started. Will be finished next week. I had to improve my PCR^2 concentration. PCR was redone. Gel will be completed next week as well.
Week 7:
101612 - Shane, go ahead and try cloning - but also start working on making more PCR^2 so that you can get a higher Nanodrop conc of your gene. Good job on the PCR's though - Dr. B
Virtual Screening Refresher was also started. The first overnight run was submitted. Second run will be submitted and analysis will be complete by Tuesday morning.
Fig. 1 PCR cleanup Nanodrop graph concenetration
After the PCR cleanup, the concentration of my target resulted in 18 ng/uL. This is slightly low, and in order to make up for this, I will add an extra volume. The 260/280 value was 1.69 and my 260/230 value was .94.
Fig. 2: PCR^2 gel electrophoresis
Lane 1: 100 bp ladder
Lane 2-5: My PCR^2 mix
This gel electrophoresis shows that my target was made and had successfully gone through the PCR reaction. Thus, all my bands match up at the designated area.
Week 6:
Fig. 1 Secondary PCR gel
Lane 1: 100 bp ladder
Lane 2: My PCR mix
Lane 3: Mihir's PCR mix
Lane 4: Akhilesh's PCR mix
The mix consisted of a reaction buffer, Hod Start Polymerase, MgSO4, 1 ul of a mix of primers (22 primers LmP, 78 ul of nanopure water), and dNTP.
My PCR mix on the gel showed up as a variety of bands. This is probably due to the primers forming the heavier strands on the top and not completing the bands on the bottom.
Week 5: Sept. 24-28
100912 - Shane - do you have your updated gels?
Our secondary PCR failed.
Fig. 1: Gel electrophoresis of secondary PCR. 1st lane was the ladder. 2nd was Sample C. 3rd was sample B. 4th was sample A. 5th was sample D (control).
10012 - after you do PCR #2, you can move ahead to cloning. Also, show your Primers for primer design. I don't know what to order here. -- Dr. B
--why is there a * star in your FASTA sequence here?
VIRTUAL PLASMID FOR TARGET: Design Primer Protocl Results:
PCR 1 was completed. The purpose of the PCR was to amplify the purple protein sequence in pGBR-22 plasmid by using M13 Reverse and Forward primers.
Figure 1: Lane 1 is the 100 bp ladder, lane 2-5 are solutions A, B, C, D, respectively.
In lane 5 however, which was the control, there is a band, which represents contamination of DNA, or DNA spilling over from one of the wells during the electrophoresis.
Week 4:
Shane - good on your purified DNA. - Dr. B
On Tuesday, September 18th, we completed filtering out the DNA from our bacteria. This completed the collection of out pNIC-Bsa4. We then checked the concentration of the plasmid using a Nanodrop procedure.
Fig. 1: The concentration of my collected plasmid is shown above. The concentration at 230 nm was about 146 ng/uL
Week 3:
-- Good gel analysis - move your legend for the gel to underneath the image. Also mention the other procedures ( Transformation, etc.) -- Dr. B 091812
This week, I worked on a restriction enzyme digest. The enzymes involved were EcoR1 and Pvu-II. We used the NEB Buffer-2 (New England Bio Labs). The first lane was skipped, the second a ladder, third the uncut plasmid which was pgbr-22, the fourth was pgbr-22 cut by Eco-R1, the fifth was pgbr-22 cut by Pvu-II, and the sixth was pgbr-22 cut by both enzymes.
Figure 1: Due to the larger bands travelling slowly, the more fluorescent molecules show up much brighter. The fourth lane was only supposed to have one cut, the fifth was supposed to have two, and the sixth was supposed to have three. However, it seems that the Pvu-II didn't completely cut the plasmid due to there being three portions shown.
We worked on a nanodrop procedure after the electrophoresis. We then began our PCR#1.
Week 2:
This week we diluted primers.
We also determined which gene was present in pNIC Bsa4.
*Background/Disease Information: The parasite is an intracellular pathogen of the immune system targeting macrophages and dendritic cells. The disease Leishmaniasis affects the populations of 88 counties worldwide with symptoms ranging from disfiguring cutaneous and muco-cutaneous lesions that can cause widespread destruction of mucous membranes to visceral disease affecting the haemopoetic organs.
Essentiality of this protein: Gene/Ortholog: Tb927.7.5160 (OG4_25847); Phenotype: significant loss of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford
The knowledge that dUTPase is an essential enzyme and that the all α-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric β-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding.
removes dUTP from the nucleotide triphosphate pool and therefore prevents the incorporation of uracil into the DNA, essential for replication and cell survival
Purification: The enzyme has proved to be a dimer by gel filtration and is able to hydrolyse both dUTP and dUDP quite efficiently, acting as a dUTP nucleotidohydrolase (dUTPase)-dUDP nucleotidohydrolase but has a limited capacity to act upon other nucleoside di- or triphosphates.
*length of your protein in Amino Acids: 268
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 30353.6
Molar Extinction coefficient of your protein at 280 nm wavelength: Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 53650 Abs 0.1% (=1 g/l) 1.768, assuming all pairs of Cys residues form cystines
Ext. coefficient 53400 Abs 0.1% (=1 g/l) 1.759, assuming all Cys residues are reduced code
Week 15:
Gen analysis was completed. Enzyme assay was also completed.
CB-kinUT was also screened. Results are as posted:
Fig. 1: Top 20 compounds with top scores from GOLD. Screening of PTPB.
Fig. 2: Enzyme Assay results. The largest absorbance reading was at an enzyme concentration of 0.63. ,
Week 14:
Virtual Screening was edited and re run on the CB306 library. Purification protocol was also completed.
Week 13:
Good, but double check these scores - you might have a column transposed - Dr. B 11/26/12Surrogate cloning was also completed with Joey's protein in mind. Bacteria cells grown were also sonicated. Next week, the protein will be harvested and a protein gel check will be used to confirm the correct protein was found and that it had not dimerized.
Week 12:
Good - Dr. B 11/19/12Week 11:
After a few attempts to get the PNIC-Bsa4 at a high concentration, cloning was attempted. Only one colony of the bacteria grew after one day, but I will try cloning again to grow more colonies.
Fig. 1: DH5alpha bacteria with gene of interest plated with Kanamycin and 5% sucrose.
Fig. 2: GEL check of pNIC-Bsa4 cut with T4 polymerase
Week 10:
This week I performed several MIDI preps for the pNIC-Bsa4 to yield a higher concentration in order to continue the cloning process. However, my first MIDI prep resulted in a low concentration due to someone switching the caps on the elution and wash buffer solution. My next MIDI prep resulted in a decent yield, so next week I will try to finish the cloning process using the 69.4 ng/uL concentration of pNIC-Bsa4. I also completed a PCR cleanup on the pNIC-Bsa4, but that only boosted my concentration by 15 ng/uL.
Week 9:
This low concentration shows that the procedure needs to be redone.
Since there were no large concentrations of the pNIC-Bsa4, new preparation using MIDI prep has to be completed. This will hopefully increase the concentration of the cut pNIC-Bsa4. From there, I can continue the cloning process.
Week 8:
102112 - good -- Dr. BVirtual Screening Refresher was completed:
Table 1: Table of Top 10 Ligands through the use of GOLD.
Cloning was also started. Will be finished next week. I had to improve my PCR^2 concentration. PCR was redone. Gel will be completed next week as well.
Week 7:
101612 - Shane, go ahead and try cloning - but also start working on making more PCR^2 so that you can get a higher Nanodrop conc of your gene. Good job on the PCR's though - Dr. BVirtual Screening Refresher was also started. The first overnight run was submitted. Second run will be submitted and analysis will be complete by Tuesday morning.
After the PCR cleanup, the concentration of my target resulted in 18 ng/uL. This is slightly low, and in order to make up for this, I will add an extra volume. The 260/280 value was 1.69 and my 260/230 value was .94.
Lane 1: 100 bp ladder
Lane 2-5: My PCR^2 mix
This gel electrophoresis shows that my target was made and had successfully gone through the PCR reaction. Thus, all my bands match up at the designated area.
Week 6:
Lane 1: 100 bp ladder
Lane 2: My PCR mix
Lane 3: Mihir's PCR mix
Lane 4: Akhilesh's PCR mix
The mix consisted of a reaction buffer, Hod Start Polymerase, MgSO4, 1 ul of a mix of primers (22 primers LmP, 78 ul of nanopure water), and dNTP.
My PCR mix on the gel showed up as a variety of bands. This is probably due to the primers forming the heavier strands on the top and not completing the bands on the bottom.
Week 5: Sept. 24-28
100912 - Shane - do you have your updated gels?Our secondary PCR failed.
10012 - after you do PCR #2, you can move ahead to cloning. Also, show your Primers for primer design. I don't know what to order here. -- Dr. B
--why is there a * star in your FASTA sequence here?
VIRTUAL PLASMID FOR TARGET:
Design Primer Protocl Results:
Upstream: TACTTCCAATCCATGAAAAACTGGGTTAAAG
Forward:
Mg++ 0
Downstream: TATCCACCTTTACTGTTAGTACAGGTATGCTTTT
Reverse Complement of the reverse primer: AAAAGCATACCTGTACTAACAGTAAAGGTGGATA
Mg++ 0
FASTA:
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTT
TAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACC
TGTACTTCCAATCCATGAAAAACTGGGTTAAAGTTACCGGTGCGGGCGTTCTCTCTGCTACTCTGCTGCTCGGT
GGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCAAACGTCAAGACCGAACAGACCCTG
AAGCCGGGTAGCCAGATCAAACTGGAAGGTGCCGTTAATGTTCGCGACCTGGGTGGTTAC
AAGACTACCGATGGTCTGACCATCAAACCGCACAAACTGATCCGCTCTGCGGAACTCGCA
AACCTCTCTGACTCTGACAAAAAGAAGCTGGTTAACACCTACGACCTGTCTCACATCGTT
GACTTCCGCACGTCTTCTGAAGTTGCGACCAAACCGGACCCGAAACTGACCGACGTGGAC
TACACCCACGACTCTGTTATGAAAGACAACGGTACGTCTACCTCTACCCAGGACCTGACG
GCGTCTCTGGCCAAGATGGACAACCCGGAAACCTTCCTGATTAACGCGAATAAATCCTTC
ATTACCGACGAGACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTGGCGAAC
CAGGACGGTTCCGTTCTGTGGCACTGCACCGCCGGTAAAGACCGTGCCGGTTTCGGCACC
GCGCTGGTTCTGTCTGCGCTGGGTGTTGATAAAAACACCGTTATCGACGACTACATGCTG
TCTAACAAATACCGTGCTGACGAAAATAAGAAAGCGATCGAAGCGGTAGCGGCGAAAACC
GACAACAAAAAAGTGATCGACGGTATGACGGCAGTTATGGAAGTTCGTGAATCTTACATC
AACGCGGCGTTCGATGAGATCAATGCGAAATATGGTTCTATGGATAACTTCCTCAAAGAA
AAGCTGGGCCTCACTGATGCGAAGAAAGAGCAGCTGAAAATAGGTGGAAATGAC AAA GCA TAC CTG TAC TAA CAG TAA AGG TGG ATA
CGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCC
GGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCC
CTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATG
GGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTT
GCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCC
GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAA
ACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTG
GAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATT
CTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATT
TAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGA
ACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAAC
TCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCC
GTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGC
GATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAG
AAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTT
CAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTG
CGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGG
CGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATG
CTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGT
CGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTA
CCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTG
ATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGG
CCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGAC
AGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA
AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC
CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG
CAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA
GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTC
TTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTG
CACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGC
GCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA
CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGA
GCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA
CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA
ACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT
GAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGC
ATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCG
CTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTC
TGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACC
GTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATG
TCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGC
GGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCAT
GGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGG
TTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGG
GTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCA
GATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAG
ACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCG
GTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGAT
CATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGA
CCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCG
CGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCAT
GATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGG
TTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGT
TGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGC
GGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTG
ATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGA
AAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTA
CCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATC
GTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGAC
ATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGC
CAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAA
TGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTC
TGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGT
CATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACA
GGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTA
ATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACT
GTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTT
TTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCG
GCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGC
GCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTAT
GCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGG
TGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAA
GCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAA
CCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCG
AAAT
PCR 1 was completed. The purpose of the PCR was to amplify the purple protein sequence in pGBR-22 plasmid by using M13 Reverse and Forward primers.
In lane 5 however, which was the control, there is a band, which represents contamination of DNA, or DNA spilling over from one of the wells during the electrophoresis.
Week 4:
Shane - good on your purified DNA. - Dr. BOn Tuesday, September 18th, we completed filtering out the DNA from our bacteria. This completed the collection of out pNIC-Bsa4. We then checked the concentration of the plasmid using a Nanodrop procedure.
Week 3:
-- Good gel analysis - move your legend for the gel to underneath the image. Also mention the other procedures ( Transformation, etc.) -- Dr. B 091812This week, I worked on a restriction enzyme digest. The enzymes involved were EcoR1 and Pvu-II. We used the NEB Buffer-2 (New England Bio Labs). The first lane was skipped, the second a ladder, third the uncut plasmid which was pgbr-22, the fourth was pgbr-22 cut by Eco-R1, the fifth was pgbr-22 cut by Pvu-II, and the sixth was pgbr-22 cut by both enzymes.
Figure 1: Due to the larger bands travelling slowly, the more fluorescent molecules show up much brighter. The fourth lane was only supposed to have one cut, the fifth was supposed to have two, and the sixth was supposed to have three. However, it seems that the Pvu-II didn't completely cut the plasmid due to there being three portions shown.
We worked on a nanodrop procedure after the electrophoresis. We then began our PCR#1.
Week 2:
This week we diluted primers.
We also determined which gene was present in pNIC Bsa4.
pLIC-forward:
NNNNNNNNNNNNNNNNACTTTAGANGGAGANNTACATATGCACCATCATCATCATCATTCTTCTGGTGT
AGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGGAGACCGACGTCCACATATACCTGCCGTTCACTAT
TATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAAC
AGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGC
AAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAAT
AGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAG
GGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCC
ATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGA
ACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTC
AAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCAT
GATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATAAAGTTCCTGAGTTCGATTCGTCCACAAT
TAAAAATATCTCTTCTGCAAAAGGNCTGGACGTTTGGGACAGCTGGCCATTACNAAACACTGACGGCACTG
TCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGA
TTTACNTGTTCTATCAAAAAGTCNGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCGCGTCTTTAAA
GACAGCGANAAATTCGATGCNAATGANTCTATCCTAAANNANNNAANNNNAGAATGGNCNGGTTCANCN
NCATTTACNTCTGANGNAAAATCCNTTNNTNCNNNNNCTGATTNNNNNCNGNNANNNNTACNGCAANN
NANNNCTGANNANNNCNNNNNNNNNATCNNNNNNNNANNGNNNNTNNANNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNTNNNNNNNNNNAANNNNNNNNNANNNNNNNNNNNTNNNNNNNNNNNNAANN
GNNNNNNNNNNNN
pLIC-reverse
NNNNNNNNNNNNNNNNNNNNTNNGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACG
GAGCTCGAATTCGGATCCGTATCCACCTTTACTGGAGACCGTCAATGCCAATAGGATATCGGCATTTTCTTT
TGCGTTTTTATTTGTTAACTGTTAATTGTCCTTGTTCAAGGATGCTGTCTTTGACAACAGATGTTTTCTTGC
CTTTGATGTTCAGCAGGAAGCTAGGCGCAAACGTTGATTGTTTGTCTGCGTAGAATCCTCTGTTTGTCATAT
AGCTTGTAATCACGACATTGTTTCCTTTCGCTTGAGGTACAGCGAAGTGTGAGTAAGTAAAGGTTACATCG
TTAGGATCAAGATCCATTTTTAACACAAGGCCAGTTTTGTTCAGCGGCTTGTATGGGCCAGTTAAAGAATT
AGAAACATAACCAAGCATGTAAATATCGTTAGACGTAATGCCGTCAATCGTCATTTTTGATCCGCGGGAGTC
AGTGAACAGGTACCATTTGCCGTTCATTTTAAAGACGTTCGCGCGTTCAATTTCATCTGTTACTGTGTTAGA
TGCAATCAGCGGTTTCATCACTTTTTTCAGTGTGTAATCATCGTTTAGCTCAATCATACCGAGAGCGCCGTT
TGCTAACTCAGCCGTGCGTTTTTTATCGCTTTGCAGAAGTTTTTGACTTTCTTGACGGAAGAATGATGTGCT
TTTGCCATAGTATGCTTTGTTAAATAAAGATTCTTCGCCTTGGTAGCCATCTTCAGTTCCAGTGTTTGCTTC
AAATACTAAGTATTTGTGGCCTTTATCTTCTACGTAGTGAGGATCTCTCAGCGTATGGTTGTCGCCTGAGCT
GTAGTTGCCTTCATCGATGAACTGCTGTACATTTTGATACNTTTTTCCGTCACCGTCAAAGATTGATTTATA
ATCCTCTACACCGTTGATGTTCAAAGAGCTGTCTGATGCTGATACGTTAACTTGTGCAGTTGTCAGTGTTTG
TTTGCCGTAANGNTTACCGNANAAATCANNGNANAATNAACNNATTTTTNNTCNGANGTAANNNNNGNT
GANNNGANNNTNCNTGNGNTTNGNCTTTNANNANNNNNCNTTNGCATCNNNTNNNCGCNNNNNTNNN
NANNNNNNNNNNTNNNGCNNNCNNNNNNNNNNCNANNTTNNNNNNNNNNNNNNNCNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCNNN
We also grew a DH5a strain of bacteria on SOC media.
In order to determine the DNA sequence of a plasmid, we made several comparisons. We then were able to determine the entire sequence for pGBR22.
Week 1:
Leishmania major
*Target (protein/gene name): deoxyuridine triphosphatase
NCBI Gene # or RefSeq#: 13391737
REFSEQ# : 157864228
*Protein ID (NP or XP #) or Wolbachia#: 29483
*Organism: Leishmania major
*Background/Disease Information: The parasite is an intracellular pathogen of the immune system targeting macrophages and dendritic cells. The disease Leishmaniasis affects the populations of 88 counties worldwide with symptoms ranging from disfiguring cutaneous and muco-cutaneous lesions that can cause widespread destruction of mucous membranes to visceral disease affecting the haemopoetic organs.
Essentiality of this protein: Gene/Ortholog: Tb927.7.5160 (OG4_25847); Phenotype: significant loss of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford
The knowledge that dUTPase is an essential enzyme and that the all α-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric β-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding.
removes dUTP from the nucleotide triphosphate pool and therefore prevents the incorporation of uracil into the DNA, essential for replication and cell survival
*EC#:3.6.1.23
Link to BRENDA EC# page: http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.6.1.23&Suchword=&organism%5B%5D=Leishmania+major&show_tm=0
Figure 1: Reaction by dUTP diphosphatase
Enzyme Assay information
-- link to paper that contains assay information
http://www.brenda-enzymes.info/literature/lit.php4?e=3.6.1.23&r=667783
ITC assay - don't have in VDS lab. Or pyrophosphate - less easy vs. phosphate. --Dr. B
-- List cost and quantity of substrate reagents and supplier
GenScript: $282.45 Full Length Sequence
-- PDB # or closest PDB entry if using homology model: 2YAY
Current Inhibitors: Calcium and DUP
Image of protein (PyMol or etc):
Figure 2: Image of dUTP from PyMOL.
*Amino Acid Sequence:
MKRARSANIP GAILHSLAEL QDGLNAMIDP SWRAVRSLDN WALAITMEST ELLDSYPWKW
WKNLNATPDL ANVRIELVDI FHFSLSGAMQ MRSTPDDEIP AASLKPLKEV MTTFLPAKEC
TSDPYGFVFF PLTDTQNAIA SFRNIIQLAN AYRFDVIIEC IIYAAEDLGF NLVAYYIAKH
TLNCIRQLSG YKDGSYVKVN NGVEDNSLLH NCIKDVSLDE VLDADKYVQA WNSIMANVYE
AFQIKESDRK DAERWFALAK ENRLAIKA
Purification:
The enzyme has proved to be a dimer by gel filtration and is able to hydrolyse both dUTP and dUDP quite efficiently, acting as a dUTP nucleotidohydrolase (dUTPase)-dUDP nucleotidohydrolase but has a limited capacity to act upon other nucleoside di- or triphosphates.
*length of your protein in Amino Acids: 268
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 30353.6
Molar Extinction coefficient of your protein at 280 nm wavelength: Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 53650
Abs 0.1% (=1 g/l) 1.768, assuming all pairs of Cys residues form cystines
Ext. coefficient 53400
Abs 0.1% (=1 g/l) 1.759, assuming all Cys residues are reduced
code
code
*CDS Gene Sequence
1 ATGAAACGTGCGCGTAGCGCGAACATCCCGGGTGCCATCCTGCACTCTCTCGCGGAGCTG
61 CAGGACGGTCTCAACGCGATGATCGACCCGTCTTGGCGTGCGGTTCGTTCTCTCGACAAC
121 TGGGCCCTCGCGATCACCATGGAATCTACCGAACTGCTCGACTCCTACCCGTGGAAGTGG
181 TGGAAGAATCTGAACGCTACCCCGGACCTGGCCAATGTGCGTATCGAACTGGTTGACATC
241 TTCCATTTCTCTCTGTCTGGTGCGATGCAGATGCGTAGCACTCCGGACGATGAAATTCCA
301 GCCGCGTCCCTGAAACCGCTGAAAGAAGTTATGACCACCTTCCTGCCGGCGAAAGAATGC
361 ACCTCTGACCCGTATGGTTTCGTTTTTTTCCCGCTCACCGACACCCAGAACGCAATCGCA
421 TCTTTCCGTAATATCATCCAGCTCGCGAATGCCTACCGTTTCGACGTTATCATCGAGTGC
481 ATCATCTACGCAGCTGAAGACCTGGGTTTCAACCTGGTTGCGTACTACATCGCGAAACAC
541 ACCCTGAACTGCATCCGTCAGCTCTCTGGTTACAAAGACGGTTCTTACGTTAAAGTTAAC
601 AACGGTGTTGAAGACAACTCTCTGCTGCACAATTGCATTAAAGATGTTTCTCTGGACGAG
661 GTCCTCGACGCGGACAAATACGTTCAGGCGTGGAACTCTATCATGGCGAACGTCTACGAA
721 GCGTTCCAGATCAAAGAGTCTGACCGTAAAGACGCAGAACGTTGGTTTGCGCTGGCGAAG
781 GAAAACCGCCTGGCGATCAAGGCGTAA
Figure 3: TMpred output for dUTP indicating the protein as not a membrane bound protein.